Loss of kinesin-8 improves the robustness of the self-assembled spindle

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants where the contribution of the spindle pole body (SPB, the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bonafide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences: for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.

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A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0497 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3647-3659 ◽

Cited By ~ 23

Author(s):

Masashi Yukawa ◽

Tomoki Kawakami ◽

Masaki Okazaki ◽

Kazunori Kume ◽

Ngang Heok Tang ◽

...

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Temperature Sensitive ◽

Bipolar Spindle ◽

Pole Body ◽

Cross Linker ◽

Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

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Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.02844.x ◽

2002 ◽

Vol 43 (6) ◽

pp. 1431-1443 ◽

Cited By ~ 14

Author(s):

Ivan Le Masson ◽

Cosmin Saveanu ◽

Anne Chevalier ◽

Abdelkader Namane ◽

Renee Gobin ◽

...

Keyword(s):

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body

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The half-bridge component Kar1 promotes centrosome separation and duplication during budding yeast meiosis

Molecular Biology of the Cell ◽

10.1091/mbc.e18-03-0163 ◽

2018 ◽

Vol 29 (15) ◽

pp. 1798-1810

Author(s):

Meenakshi Agarwal ◽

Hui Jin ◽

Melainia McClain ◽

Jinbo Fan ◽

Bailey A. Koch ◽

...

Keyword(s):

Chromosome Segregation ◽

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body ◽

Centrosome Separation ◽

Chromatid Segregation ◽

Yeast Meiosis ◽

Unique Mechanism

The budding yeast centrosome, often called the spindle pole body (SPB), nucleates microtubules for chromosome segregation during cell division. An appendage, called the half bridge, attaches to one side of the SPB and regulates SPB duplication and separation. Like DNA, the SPB is duplicated only once per cell cycle. During meiosis, however, after one round of DNA replication, two rounds of SPB duplication and separation are coupled with homologue segregation in meiosis I and sister-chromatid segregation in meiosis II. How SPB duplication and separation are regulated during meiosis remains to be elucidated, and whether regulation in meiosis differs from that in mitosis is unclear. Here we show that overproduction of the half-bridge component Kar1 leads to premature SPB separation during meiosis. Furthermore, excessive Kar1 induces SPB overduplication to form supernumerary SPBs, leading to chromosome missegregation and erroneous ascospore formation. Kar1-mediated SPB duplication bypasses the requirement of dephosphorylation of Sfi1, another half-bridge component previously identified as a licensing factor. Our results therefore reveal an unexpected role of Kar1 in licensing meiotic SPB duplication and suggest a unique mechanism of SPB regulation during budding yeast meiosis.

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Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Molecular Biology of the Cell ◽

10.1091/mbc.e18-04-0261 ◽

2018 ◽

Vol 29 (18) ◽

pp. 2176-2189 ◽

Cited By ~ 1

Author(s):

Christine M. Jones ◽

Jun-Song Chen ◽

Alyssa E. Johnson ◽

Zachary C. Elmore ◽

Sierra N. Cullati ◽

...

Keyword(s):

Cell Division ◽

Chromosome Segregation ◽

Ubiquitin Ligase ◽

Spindle Pole Body ◽

Mitotic Checkpoint ◽

Spindle Pole ◽

Checkpoint Activation ◽

Pole Body ◽

Septation Initiation Network ◽

Anaphase B

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

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Mps1p Regulates Meiotic Spindle Pole Body Duplication in Addition to Having Novel Roles during Sporulation

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3525 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3525-3537 ◽

Cited By ~ 27

Author(s):

Paul D. Straight ◽

Thomas H. Giddings ◽

Mark Winey

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Pole Body ◽

Class Ii ◽

Spindle Pole ◽

Spore Wall ◽

Meiotic Spindle ◽

Restrictive Temperature ◽

Wall Formation ◽

Pole Body

Sporulation in yeast requires that a modified form of chromosome segregation be coupled to the development of a specialized cell type, a process akin to gametogenesis. Mps1p is a dual-specificity protein kinase essential for spindle pole body (SPB) duplication and required for the spindle assembly checkpoint in mitotically dividing cells. Four conditional mutant alleles of MPS1disrupt sporulation, producing two distinct phenotypic classes. Class I alleles of mps1 prevent SPB duplication at the restrictive temperature without affecting premeiotic DNA synthesis and recombination. Class II MPS1 alleles progress through both meiotic divisions in 30–50% of the population, but the asci are incapable of forming mature spores. Although mutations in many other genes block spore wall formation, the cells produce viable haploid progeny, whereas mps1 class II spores are unable to germinate. We have used fluorescently marked chromosomes to demonstrate that mps1 mutant cells have a dramatically increased frequency of chromosome missegregation, suggesting that loss of viability is due to a defect in spindle function. Overall, our cytological data suggest that MPS1 is required for meiotic SPB duplication, chromosome segregation, and spore wall formation.

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The budding yeast RSC complex maintains ploidy by promoting spindle pole body insertion

The Journal of Cell Biology ◽

10.1083/jcb.201709009 ◽

2018 ◽

Vol 217 (7) ◽

pp. 2445-2462 ◽

Cited By ~ 4

Author(s):

Tina L. Sing ◽

Minnie P. Hung ◽

Shinsuke Ohnuki ◽

Godai Suzuki ◽

Bryan-Joseph San Luis ◽

...

Keyword(s):

Chromosome Segregation ◽

Cell Function ◽

Spindle Pole Body ◽

Spindle Pole ◽

Eukaryotic Cells ◽

Pole Body ◽

Rapid Transition ◽

Rsc Complex ◽

Diagnostic Changes ◽

Insight Into

Ploidy is tightly regulated in eukaryotic cells and is critical for cell function and survival. Cells coordinate multiple pathways to ensure replicated DNA is segregated accurately to prevent abnormal changes in chromosome number. In this study, we characterize an unanticipated role for the Saccharomyces cerevisiae “remodels the structure of chromatin” (RSC) complex in ploidy maintenance. We show that deletion of any of six nonessential RSC genes causes a rapid transition from haploid to diploid DNA content because of nondisjunction events. Diploidization is accompanied by diagnostic changes in cell morphology and is stably maintained without further ploidy increases. We find that RSC promotes chromosome segregation by facilitating spindle pole body (SPB) duplication. More specifically, RSC plays a role in distributing two SPB insertion factors, Nbp1 and Ndc1, to the new SPB. Thus, we provide insight into a role for a SWI/SNF family complex in SPB duplication and ploidy maintenance.

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Inactivation of Mitotic Kinase Triggers Translocation of MEN Components to Mother-Daughter Neck in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e03-04-0238 ◽

2003 ◽

Vol 14 (11) ◽

pp. 4734-4743 ◽

Cited By ~ 31

Author(s):

Hong Hwa Lim ◽

Foong May Yeong ◽

Uttam Surana

Keyword(s):

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Pole ◽

Mitotic Exit ◽

Mitotic Kinase ◽

Division Site ◽

Pole Body ◽

Spindle Pole Bodies ◽

Mitotic Exit Network ◽

Mutant Cells

Chromosome segregation, mitotic exit, and cytokinesis are executed in this order during mitosis. Although a scheme coordinating sister chromatid separation and initiation of mitotic exit has been proposed, the mechanism that temporally links the onset of cytokinesis to mitotic exit is not known. Exit from mitosis is regulated by the mitotic exit network (MEN), which includes a GTPase (Tem1) and various kinases (Cdc15, Cdc5, Dbf2, and Dbf20). Here, we show that Dbf2 and Dbf20 functions are necessary for the execution of cytokinesis. Relocalization of these proteins from spindle pole bodies to mother daughter neck seems to be necessary for this role because cdc15-2 mutant cells, though capable of exiting mitosis at semipermissive temperature, are unable to localize Dbf2 (and Dbf20) to the “neck” and fail to undergo cytokinesis. These cells can assemble and constrict the actomyosin ring normally but are incapable of forming a septum, suggesting that MEN components are critical for the initiation of septum formation. Interestingly, the spindle pole body to neck translocation of Dbf2 and Dbf20 is triggered by the inactivation of mitotic kinase. The requirement of kinase inactivation for translocation of MEN components to the division site thus provides a mechanism that renders mitotic exit a prerequisite for cytokinesis.

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NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.121.3.503 ◽

1993 ◽

Vol 121 (3) ◽

pp. 503-512 ◽

Cited By ~ 162

Author(s):

P Y Goh ◽

J V Kilmartin

Keyword(s):

Chromosome Segregation ◽

Staining Pattern ◽

Essential Gene ◽

Spindle Pole Body ◽

Spindle Pole ◽

Polyploid Cell ◽

Temperature Sensitive ◽

Sensitive Mutant ◽

Pole Body

A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.

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Dissociation of the Nuf2-Ndc80 Complex Releases Centromeres from the Spindle-Pole Body during Meiotic Prophase in Fission Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-0996 ◽

2005 ◽

Vol 16 (5) ◽

pp. 2325-2338 ◽

Cited By ~ 48

Author(s):

Haruhiko Asakawa ◽

Aki Hayashi ◽

Tokuko Haraguchi ◽

Yasushi Hiraoka

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Meiotic Prophase ◽

Spindle Pole Body ◽

Spindle Pole ◽

Mating Pheromone ◽

Pole Body ◽

Pheromone Signaling ◽

Ndc80 Complex ◽

Mutant Cells

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.

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Meiotic telomere clustering requires actin for its formation and cohesin for its resolution

The Journal of Cell Biology ◽

10.1083/jcb.200501042 ◽

2005 ◽

Vol 170 (2) ◽

pp. 213-223 ◽

Cited By ~ 103

Author(s):

Edgar Trelles-Sticken ◽

Caroline Adelfalk ◽

Josef Loidl ◽

Harry Scherthan

Keyword(s):

Chromosome Segregation ◽

Actin Polymerization ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body ◽

Telomere Cluster ◽

Cohesin Subunit ◽

Yeast Meiosis ◽

Bouquet Stage ◽

Meiosis I

In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster–SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8Δ meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization–dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.

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